One of the best studied actions of insulin is its ability to rapidly increase the rate of glucose transport, a major control point in regulation of cellular carbohydrate metabolism in adipose and muscle tissue. Our laboratory is studying the molecular mechanisms by which insulin, and hormones which antagonize the actions of insulin on carbohydrate metabolism, such as the glucocorticoids, interact to regulate the rate of glucose transport. Recent work suggests that insulin increases glucose transport at least in part by increasing the number of transporters in the plasma membrane, but may also cause structural changes in the transporter. Using [3H]cytochalasin B to affinity-label the glucose transporter, we have found that glucocorticoids appear to decrease the number of transporters in the plasma membrane. This grant will address the question of whether glucocorticoids and insulin alter both the number and structure of glucose transporters in rat adipocyte plasma membranes. Cytochalasin B binding and affinity-labeling will be used to examine whether glucocorticoids cause a decrease in the number of transporters in plasma membranes. The ability of glucocorticoids to cause transporters to be degraded or inactivated, or to be redistributed into intracellular membranes will be examined. We will also test whether glucocorticoids alter the number of glucose transporters by altering phospholipase A2activity. In order to test whether insulin or glucocorticoids alter the structure of glucose transporters, glucose transporters will be identified and analyzed in 2-dimensional gels by affinity-labeling or anti-transporter antibodies. To facilitate these studies, polyclonal and monoclonal antibodies to the glucose transporter will be prepared. These are greatly needed probes for analyzing the effects of insulin, glucocorticoids, and other hormones on glucose transport. We will test for alterations in charge, size, state of phosphorylation and carbohydrate content of the glucose transporter, before and after hormone addition, and in the different membrane fractions. The antibodies will also be used to purify the glucose transporter to permit additional structural studies. These studies will provide a) new and important information concerning the cellular actions of both insulin and the glucocorticoids; b) structural information about the rat adipocyte glucose transporter; c) important new probes (antibodies) for studying the regulation of glucose transport; as well as d) insight into the mechanisms by which insulin resistance occurs.